Enzyme Catalysis and Regulation
Structural and Kinetic Evidence for an Extended Hydrogen-bonding Network in Catalysis of Methyl Group Transfer: ROLE OF AN ACTIVE SITE ASPARAGINE RESIDUE IN ACTIVATION OF METHYL TRANSFER BY METHYLTRANSFERASES*

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The methyltetrahydrofolate (CH3-H4folate) corrinoid-iron-sulfur protein (CFeSP) methyltransferase (MeTr) catalyzes transfer of the methyl group of CH3-H4folate to cob(I)amide. This key step in anaerobic CO and CO2 fixation is similar to the first half-reaction in the mechanisms of other cobalamin-dependent methyltransferases. Methyl transfer requires electrophilic activation of the methyl group of CH3-H4folate, which includes proton transfer to the N5 group of the pterin ring and poises the methyl group for reaction with the Co(I) nucleophile. The structure of the binary CH3-H4folate/MeTr complex (revealed here) lacks any obvious proton donor near the N5 group. Instead, an Asn residue and water molecules are found within H-bonding distance of N5. Structural and kinetic experiments described here are consistent with the involvement of an extended H-bonding network in proton transfer to N5 of the folate that includes an Asn (Asn-199 in MeTr), a conserved Asp (Asp-160), and a water molecule. This situation is reminiscent of purine nucleoside phosphorylase, which involves protonation of the purine N7 in the transition state and is accomplished by an extended H-bond network that includes water molecules, a Glu residue, and an Asn residue (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Shi, W., Fedorov, A., Lewandowicz, A., Cahill, S. M., Almo, S. C., and Schramm, V. L. (2002) Biochemistry 41, 14489-14498). In MeTr, the Asn residue swings from a distant position to within H-bonding distance of the N5 atom upon CH3-H4folate binding. An N199A variant exhibits only ∼20-fold weakened affinity for CH3-H4folate but a much more marked 20,000-40,000-fold effect on catalysis, suggesting that Asn-199 plays an important role in stabilizing a transition state or high energy intermediate for methyl transfer.

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This paper is dedicated to Professor Martha Ludwig, who passed away on November 27, 2006. Martha was a thoughtful, thorough, and innovative scientist who made extremely important contributions to our understanding of the structure and function of cobalamin-dependent methyltransferases. Martha was a crystallographer who was a faculty member in the Biological Chemistry Department at the University of Michigan for almost 40 years and a member of the National Academy of Sciences and of the Institute of Medicine. She also was a caring friend who we wish could have seen this paper in print.

The atomic coordinates and structure factors (codes 2E7F and 2OGY) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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This article was selected as a Paper of the Week.

1

Both authors contributed equally to the publication (alphabetical order).

2

Present Address: Stanford Linear Accelerator Center, 2575 Sand Hill Rd., Menlo Park, CA 94025.

3

Present Address: Dept. of Applied Molecular Bioscience, Nagoya University, Graduate School of Bioagricultural Sciences, Furo-Cho, Chikusa-Ku, Nagoya 464-8601, Japan.